Method of altering the binding specificity of monoclonal antibodies by oxidation-reduction reactions

ABSTRACT

The binding specificity of a monoclonal antibody is altered by exposing the monoclonal antibody to an oxidizing agent or an electric potential.

FIELD OF INVENTION

The present invention relates to a method of reversibly altering thebinding specificity of monoclonal antibodies.

BACKGROUND OF THE INVENTION

The present inventor has previously reported the discovery that bloodand other bodily fluids from normal individuals contain a significantnumber of autoantibodies, that, when treated with an oxidizing agent,become capable of binding self antigens. See, for example, the followingpublications:

-   McIntyre, J A. “The appearance and disappearance of antiphospholipid    antibodies subsequent to oxidation-reduction reactions.” Thromb.    Res. 2004; 114:579-87.-   McIntyre, J A, Wagenknecht, D R, & Faulk, W P. “Autoantibodies    unmasked by redox reactions.” J. Autoimmun 2005; 24:311-17.-   McIntyre, J A, Wagenknecht, D R, & Faulk, W P. “Redox-reactive    autoantibodies: Detection and physiological relevance.” Autoimm.    Rev. 2006; 5:76-83.-   McIntyre, J A, Chapman, J, Shavit, E, Hamilton, R L, DeKosky, S T.    “Redox-reactive autoantibodies in Alzheimer's patients'    cerebrospinal fluids: Preliminary studies.” Autoimmunity, 2007;    40:390-6.-   McIntyre, J A, Hamilton, R L, DeKosky, S T. “Redox-reactive    autoantibodies in cererebrospinal fluids.” Ann. N.Y. Acad. Sci.    2007; 1109: 296-302.-   U.S. Patent Application Publication No. 2005/0260681 A!-   and U.S Patent Application Publication No. 2005/0101016 A1.

In these publications, it was reported that blood from normalindividuals contains a significant number of autoantibodies, in a widevariety of isotypes and specificities, but that these autoantibodiesbecome detectible only when certain body fluids or blood are exposed tooxidation, by, for example an oxidizing agent or electric current,according to a method described therein. It was reported that samplessuch as blood, plasma, serum, breast milk, cerebrospinal fluid, andpurified immunoglobulin fractions can be treated by oxidation and thenassayed with a variety of self antigens and other types of antigens toidentify masked autoantibodies that can be unmasked by oxidation.Autoantibodies that have been unmasked by oxidation include thefollowing in Table 2:

TABLE 2 Masked autoantibodies identified to date after redox conversionof normal plasma or IgG. Specificity Assay Method of Detection Glutamicacid decarboxylase (GAD) RIA Tyrosine phosphatase (IA-2) RIAAntiphospholipid antibodies: ELISA aPS, aPE, aCL, aPC Lupusanticoagulant (LA) APTT, dRVVT Antinuclear antibodies (ANA) RELISA ®Anti-nucleolus immunofluorescence Anti-lamin, nuclear membranesimmunofluorescence Anti-mitochondria immunofluorescence Anti-Golgiimmunofluorescence Anti-granulocyte, neutrophil, Flow Cytometry (FACS)monocyte Anti-B lymphocytes FACS Anti-myeloperoxidase ELISA Anti-tumorcell lines Western blot Anti-trophoblast immunofluorescence Anti-factorVIII ELISA Platelet factor 4/heparin complex ELISAAnti-beta2-glycoprotein I ELISA Red Blood cells Ortho Gel Cards Ro/SS-AELISA Anti-human antigens* Invitrogen ProtoArray ® Table 2 abbreviationsused: aCL, anticardiolipin aPC, antiphosphatidylcholine aPE,antiphosphatidylethanolamine aPS, antiphosphatidylserine APPT, activatedpartial thromboplastin time dRVVT, dilute Russell's viper venom timeELISA, enzyme-linked immunosorbant assay RIA, radioimmunoassay*5,000-6,000 of 8,000 human antigens tested by microarray are recognizedby redox-sensitive autoantibodies.

It has now been discovered that the binding specificity of monoclonalantibodies can be altered by similar treatments with an oxidizing agentor a direct electric current. This finding is significant, sincemonoclonal antibodies are typically intended to bind only a specificantigen. However, according to the method described herein, the spectrumof activity of a monoclonal antibody can be broadened to includeantigens other than the specific antigen that the monoclonal antibody isintended to bind.

SUMMARY OF THE INVENTION

According to an aspect of the present invention, there is provided amethod comprising providing a composition containing a monoclonalantibody, the monoclonal antibody having a binding specificity toward aspecific antigen, and exposing the composition to an oxidizing agent oran electric potential sufficient to effect an alteration of the bindingspecificity of the monoclonal antibody.

According to another aspect of the present invention there is provided acomposition comprising a monoclonal antibody having a bindingspecificity that has been altered by exposure of the monoclonal antibodyto an oxidizing agent or an electric potential.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the aPS, aCL, aPE, and aPC binding profiles(as measured by optical density, OD) of an antiglycophorin A monoclonalantibody in an adult bovine plasma (ABP) containing diluent buffer thatwas designated as a “control”, and in the identical buffer diluent afterexposure of the monoclonal antibody to an electric potential that wasdesignated as “redox” according to an embodiment of the presentinvention. The same ELISA test parameters were also conducted using abovine serum albumin (BSA) containing diluent buffer that was designatedas a “control” and in the identical buffer diluent after exposure of themonoclonal antibody to an electric potential that was designated as“redox” according to an embodiment of the present invention.

FIG. 2 is a graph showing the aPS, aCL, aPE, and aPC binding profiles(as measured by OD) of a monoclonal anti-CXCR4 antibody diluted in abuffer containing ABP (control), and in an identical buffer afterexposure of the monoclonal antibody to an electric potential (redox)according to an embodiment of the present invention. Similarly themonoclonal was diluted into a buffer containing BSA (control) and intothe identical buffer after exposure of the monoclonal antibody to anelectric potential (redox) according to an embodiment of the presentinvention.

FIG. 3 is a graph showing the aPS, aCL, aPE, and aPC binding profiles(as measured by OD) of a CD 63 monoclonal antibody in a diluent buffercontaining ABP (control) and into an identical buffer after treatment ofthe monoclonal antibody with an electric potential (redox) according toan embodiment of the present invention. Similarly, the monoclonal wasdiluted into a buffer containing BSA (control) and into the identicalbuffer after exposure of the monoclonal antibody to an electricpotential (redox) according to an embodiment of the present invention.

FIG. 4 is a graph showing the amount of aPS, aCL, aPE, and aPC binding(as measured by OD) of a beta₂glycoprotein-I (β₂GP-I) monoclonalantibody in an ABP containing buffer diluent (control), and in an ABPbuffer diluent after exposure of the monoclonal antibody to an electricpotential according to an embodiment of the present invention.Similarly, the monoclonal was diluted into a buffer containing BSA(control) and into the identical buffer after exposure of the monoclonalantibody to an electric potential (redox) according to an embodiment ofthe present invention.

FIG. 5 is a graph showing the aPS, aCL, aPE, and aPC binding profiles(as measured by OD) of a monoclonal antibody to clotting factor VII.This particular monoclonal was refractory to oxidative alterations andno aPL unmasking was observed.

FIG. 6 is a graph showing the aPS, aCL, aPE, and aPC binding profiles(as measured by OD) of a monoclonal antibody to clotting factor IX.Notice that in the diluent buffer containing ABP factor IX binds to thenegatively charged phospholipids PS and CL (control), thus a positivemonoclonal anti-factor IX reaction is seen as aPS and aCL. Redoxexposure via EMF had no masking effect upon Factor IX binding in the ABPdiluent. In contrast, in the ABS diluent (control) where no factor IX ispresent, no aPL activity is observed. However, after redox exposure,both aPE and aPC activities are unmasked.

FIG. 7 is a graph showing the aPS, aCL, aPE, and aPC binding profiles(as measured by OD) of an IgG1 monoclonal antibody sold commercially asan IgG isotype control. In this graph a comparison is made of thedifferential effects of two different oxidizing agents, hemin and EMF.Details are provided in the figure legend.

FIG. 8 as in FIG. 5 is a graph showing the aPS, aCL, aPE, and aPCbinding profiles (as measured by OD) of a monoclonal antibody to the CD44 antigen. This monoclonal antibody is not significantly altered in itsbinding activity by oxidizing agent, hemin or EMF. The monoclonal does,however, continue to bind its cell surface antigen.

FIG. 9 is a graph showing the aPS, aCL, aPE, and aPC binding profiles(as measured by OD) of a monoclonal antibody to platelet antigen,IIb-IIIa. In an ABP containing dilution buffer (control) no activity isseen as aPS or aCL. After the monoclonal in suspension was treated withan electric potential (redox) according to an embodiment of the presentinvention both aPS and aCL became unmasked as shown by using this bufferdiluent. In the BSA containing buffer diluent (control) no activity toaPS or aCL was observed, thus the plasma proteins bound by thenegatively charged PS and CL in the ABP buffer diluent was not presentin the BSA containing buffer diluent. Treatment of the monoclonal withan electric potential (redox) according to an embodiment of the presentinvention caused an increase in strength of the aPE and aPC signals inthe BSA containing buffer.

FIG. 10( a) and (b) are graphs showing the effect of EMF exposure on amonoclonal antibody produced to a plasma membrane antigen found on themurine tumor cell line SP2/0. This monoclonal antibody was tailor madefor the express purpose of controlling for the proprietary variablesthat may exist in commercially prepared monoclonal antibodypreparations. For example, might the suspension solutions used bycommercial monoclonal antibody producers be contaminated with animalserum as residual from the monoclonal culture growth media? Suchcontamination could interfere with masking and/or unmasking observationsof the mouse monoclonals.

As shown in FIG. 10, the experimental design allowed for testing of theculture media by itself, the culture media containing the monoclonalantibody, the culture media after exposure to EMF and the culture mediacontaining the monoclonal antibody after EMF exposure. In addition, wehad access to the monoclonal antibody concentrated by using a protein Aaffinity column (1.46 mg/ml) for further testing. FIG. 10 shows that allaPL unmasking was detected after the monoclonal antibody was treated byEMF and this was observed whether detection was in the ABP containingdiluent buffer or the BSA containing buffer diluent.

FIG. 11 is a graph showing the effect of EMF oxidation over time usingthe monoclonal antibody anti-glycophorin A. This is the identicalmonoclonal antibody preparation that is shown in FIG. 1. FIG. 11demonstrates that during the first minute of EMF exposure, the bindingof this monoclonal to its red blood cell (RBC) target membrane antigenas measured by flow cytometry mean channel shifts (MCS), slopesdownward. However, after the first minute wherein aliquots were obtainedat 5 second intervals for flow cytometry, an uninterrupted EMF exposurefor an additional minute caused a reversal of the downward trend and anupward shift to approximate the MCS value shown to occur after theinitial 15-20 seconds of EMF exposure. A MCS shift from 401 to 386 whichwas observed at the 10 second interval and which corresponds to the timefor unmasking the aPL depicted in FIG. 1 would not be consideredsignificant by flow cytometry operators. Nonetheless, it has a profoundeffect upon the monoclonals' binding properties. Thus, the unmasking(alteration) of aPL reactivity has little effect upon the monoclonalsRBC binding properties. The extended 1 minute EMF oxidation step,however, indicates that the downward alteration of binding to RBC duringthe first minute is reversible.

The two graphs comprising FIGS. 12A and 12B show that the oxidativetreatment of the monoclonal antibodies shown also in FIG. 10 is a timeand temperature reversible alteration of unmasking and masking. Whenstored at −80° C. there is no significant loss of aPL activity after EMFtreatment. In contrast, storage at 37° C. for 4-days results in loss ofaPL reactivity, however, the aPL reactivity can be recovered (unmasked)completely by exposing the monoclonal antibody suspension to another EMFtreatment. This phenomenon could have important physiologicaldisadvantages in vivo inasmuch as oxidized therapeutic monoclonalautoantibodies could appear (unmask) in areas where reactive oxygenspecies are generated, such as in sites of inflammation. Unmasking ofthese therapeutic monoclonal autoantibodies could lead topathophysiological effects undesirable for the patient recipients.Indeed, this may be a reason why some therapeutic monoclonal antibodieshave been discontinued from use because of their untoward andunanticipated side effects.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of altering the bindingspecificity of a monoclonal antibody.

The term “altering the binding specificity” of a monoclonal antibodyrefers to a process whereby a monoclonal antibody is changed or altered,such as by oxidation and reduction, so that it becomes capable ofspecific binding of an antigen or ligand that it had not previously beencapable of specifically binding. For example, the spectrum of antibodyactivity may be broadened so that the monoclonal antibody binds to otherantigens. The term “altering the binding specificity” may also apply toa process whereby a monoclonal antibody is changed or altered, such asby oxidation or reduction so that it becomes incapable of specificbinding of an antigen or ligand that it had previously been capable ofspecifically binding, but it is to be understood that in this context,the term refers to a reversible change and not a permanent, irreversiblechange such as denaturing of the protein.

The term “monoclonal antibody” refers generally to an antibody that hasbeen selected on the basis of binding specificity towards a particularantigen and then cloned or otherwise manufactured to produce a set ofantibody molecules each having an identical molecular structure.Monoclonal antibodies for many antigens are commercially available.

In the method of the present invention, the binding specificity of amonoclonal antibody is altered by exposing the monoclonal antibody to anoxidant or to an electric current. For example, the binding specificityof a monoclonal antibody can by altered so that the monoclonal antibodyis able to bind an antigen that it was not able to bind before themethod was carried out.

If an oxidizing agent is used to carry out the method of the invention,the oxidizing agent can be any compound that is capable of altering theredox state of a biological molecule. More specifically, the oxidizingagent is a molecule that has the ability to be reduced by acting as anelectron acceptor for other molecules that act as electron donors.Suitable oxidizing agents include many compounds that contain acoordinated (transition) metal that can participate inoxidation-reduction reactions, for example a redox capable metal such asiron that can alternate between its ferric and ferrous states. Otherexamples of oxidizing agents include, but are not limited to hemin andthe coordinated magnesium metal in chlorophyll molecules, sodiumperiodate (NaIO₄) and potassium permanganate (KMnO₄). Typically, when atransitional metal oxidizing agent is used, a mixture of the monoclonalantibody and the oxidizing agent must be incubated for a period of time,typically for 12-24 hours. The oxidizing agent should be used at aconcentration sufficient to alter the binding specificity of themonoclonal antibody, but not at a concentration that might destroy ordenature the monoclonal antibody. It has been found that different typesof monoclonal antibodies can interact differently with differentantioxidants.

If a DC electric current is used to carry out the method of theinvention, the method may be carried out by any means of delivering anelectric current, such as by immersing positive and negative electrodesinto a conductive solution containing the sample to be treated. Asolution containing a monoclonal antibody is exposed to an electricpotential of a sufficient magnitude and of a sufficient duration toalter the binding specificity of the monoclonal antibody. It has beenfound that positive results may be obtained by exposing a solution to anelectric potential of 6-24 volts for a few seconds to a few minutes. Anextended exposure to an electric current may result in reversal of thealteration of the binding specificity. This has been seen by using aglycophorin A monoclonal antibody and flow cytometry and measuring theantibody's reactivity to red blood cells overtime of EMF exposure. FIG.11 shows a reduction in the mean channel shift (MCS) as EMF exposureincreases at 5 second intervals during the first 60 seconds. However,the application of an additional minute EMF exposure (2-minutes total)causes a reversal of the downward trend and an increase in the MCSvalue.

Without being bound to a specific theory, it is believed that exposureof a monoclonal antibody to the oxidizing agent or electric current canoxidize and/or reduce an antigen binding site in the Fab portion of themonoclonal antibody. It has been found in oxidation experimentsconducted with IVIg and using detection by monoclonal anti-nitrotyrosineantibodies that IgG that has been exposed to oxidation by hemin has agreater degree of nitrosylation than non-treated IgG. Accordingly, itcan be theorized that a similar mechanism might be operative withmonoclonal antibodies and that the alteration of the antigen bindingsite of the monoclonal antibody is effected by reversible nitrosylationof aromatic ring containing amino acids (e.g., tyrosine and tryptophan)in and around the antibody hypervariable region, which may produceconformational changes in the antigen binding site.

Whether a particular monoclonal antibody of interest is one that has abinding specificity that can be altered by changing its redox state andthe effectiveness of any set of conditions for altering the bindingspecificity of the monoclonal antibody of interest may be readilydetermined by subjecting the monoclonal antibody to a change in redoxstate by, for example, exposing the monoclonal antibody to an oxidizingagent or electric current and then using ELISA or other ligand-receptorassays to determine whether the binding specificity of the monoclonalantibody has been altered. In other words, an assay of a monoclonalantibody can be carried out before and after the monoclonal antibody issubjected to a change in redox conditions to see whether the process hasaltered the binding specificity of the monoclonal antibody. For example,the best oxidizing agent or method to alter a specific monoclonalantibody can be readily determined by simple experimentation.Experimental data have shown that different oxidizing agents can unmaskdifferent autoantibody specificities.

A further aspect of the present invention is a monoclonal antibody thathas been altered by exposure to an oxidizing agent or electric current.As explained more fully in the examples below, it has been found to datethat the binding specificity of the following monoclonal antibodies canbe altered to change their binding profile with respect to at least oneof cardiolipin, phosphatidylcholine, phosphatidylethanolamine andphosphatidylserine: an anti-glycophorin A monoclonal antibody; ananti-CXCR4 monoclonal antibody; a CD 63 monoclonal antibody; a B₂GP-1monoclonal antibody; an anti-platelet IIb-IIIa monoclonal antibody; anda gamma 1 mouse isotype control monoclonal antibody. Alterations inbinding properties of a monoclonal antibody to Factor VII and amonoclonal antibody to CD 44 as well as a monoclonal antibody to FactorIX were dependent upon the dilution buffers used for the ELISA testing.

EXAMPLES

Having described the invention, the following examples are given toillustrate specific applications of the invention, including the bestmode now known to perform the invention. These specific examples are notintended to limit the scope of the invention described in thisapplication.

Regarding each of the described herein, unless otherwise noted, thefollowing procedure was typically used: 250 μl of monoclonal antibodywas used directly from the vial or bottle supplied by the commercialvendor. It was placed as a bubble on a parafilm sheet. Graphiteelectrodes were connected to the positive and negative terminals of 6-9volt battery or a power source (BK Precision) set at 8-volts and weresubmersed into the bubble solution containing monoclonal antibodies for10 seconds. (Where noted herein, an alternative treatment was used inwhich the monoclonal antibody was combined with hemin and the mixturewas incubated, with rocking or shaking, at 36° C. for a period of 12-24hours.) Following the treatment with electric current or with hemin, asample of the monoclonal antibody was tested for the presence ofantiphospholipid antibodies (aPL) using a comprehensive in-house ELISAaPL format that provides separate aPL test results. The testingprocedure is described in greater detail in the following publications,incorporated herein by reference: Wagenknecht, D R, et al., TheEvolution, Evaluation and Interpretation of Antiphospholipid AntibodyAssays, Clinical Immunology Newsletter, Vol. 15, No. 2/3 (1995) pp.28-38 and McIntyre, J A, et al., Frequency and Specificities ofAntiphospholipid Antibodies (aPL) in Volunteer Blood Donors,Immunobiology 207(1): 59-63, 2003.

Four aPL specificities were assessed, 1) aPS=antiphosphatidylserine, 2)aCL=anticardiolipin, 3) aPE=antiphosphatidylethanolamine, and 4)aPC=antiphosphatidylcholine. Each monoclonal antibody sample, before andafter oxidation was diluted 1/10 into and assessed in the presence(dependent) and absence (independent) of a TRIS buffer diluentssupplemented with either 10% adult bovine plasma (ABP), which containsthe phospholipid-binding plasma proteins or 1% bovine serum albumin,(BSA, which is devoid of phospholipid-binding plasma proteins),respectively.

With the exception of FIGS. 7, 10 and 12, the monoclonal antibodies usedfor the figures shown in this application were to human proteins andprepared and sold commercially for hospital laboratory use. These murinemonoclonal antibodies represented subclasses IgG1, IgG2a and IgG2b.Thus, all IgG subclass are susceptible to oxidation-reduction (redox)alterations. Similar to published polyclonal antibody data, we detectunmasking and masking of monoclonal antibody reactivity subsequent toredox reactions. All ELISA data cited were done in triplicate. We usedan in-house aPL ELISA to test for binding alterations resulting fromoxidation-reduction reactions because we have years of experience withthis assay and our laboratory does thousands of these tests yearly.Procedural descriptions of the assay can be found in the publicationslisted above. All monoclonal antibody suspensions were diluted 1/10before testing in an ELISA unless otherwise stated.

The results in the aPL specificities obtained for the variousexperiments described herein are given in the accompanying figures. Thepositive/negative findings are expressed in terms of optical density(OD). In describing the results herein, the term “unmasking” refersgenerally to the condition in which an alteration of binding specificityof a monoclonal antibody is observed, such as, for example, where anenhanced binding to a phospholipid antigen is observed.

Example 1

A 250 μl bubble of the manufacturer's solution containing a mousemonoclonal IgG2b antibody to human glycophorin A was placed on aparafilm platform. The solution was exposed to 10 seconds of 8-volt EMFby immersing two electrodes (anode and cathode) for 10 seconds with apower source set at 8 volts. Each monoclonal antibody solution wasassayed before and after oxidation for the following bindingspecificities: antiphosphatidylserine (aPS), anticardiolipin (aCL),antiphosphatidylethanolamine (aPE), and antiphosphatidylcholine (aPC)The control and redox exposed solutions were then assayed for aPS, aCL,aPE and aPC binding specificities each diluted 1/10 into separate TRISdiluent buffers, one supplemented with 10% adult bovine plasma (ABP) andthe other supplemented with 1% bovine serum albumin (BSA). The bindingprofiles for the diluents containing ABP and BSA and theanti-glycophorin A monoclonal antibody before EMF treatment (“control”)and after EMF treatment (“redox”) are shown in FIG. 1. It can be seen inFIG. 1 that untreated, control, glycophorin A had no antiphospholipidantibody (aPL) activity, but after redox exposure by using electromotiveforce (EMF), significant aPL reactivity was detected. The ELISA for aPLdetection was performed in two diluents containing either 10% adultbovine plasma (ABP) or 1% bovine serum albumin (BSA), both in TRISbuffer. The ABP supplements the buffer with plasma proteins as certainaPL require phospholipid binding proteins to become detectable whereasaPL independent of phospholipid binding proteins will bind withoutsupplemental plasma proteins. In this example, binding to PS isequivocal in both buffers. Binding to PE is only observed in the BSAbuffer indicating that the aPE is not binding to PE in the presence ofABP because it is inhibited by a plasma protein that has a higheraffinity for binding PE than the aPE.

Example 2

Example 1 was repeated, except that a mouse anti-CXCR4 monoclonalantibody, a co-receptor for the HIV infection of CD4 positive cells, wasused as the monoclonal antibody instead of an anti-glycophorin Amonoclonal antibody. The treatment and testing format was the same asfor FIG. 1. The binding profiles for the diluents containing ABP and BSAand the anti-CXCR4 monoclonal antibody before EMF treatment (“control”)and after EMF treatment (“redox”) are shown in FIG. 2. As shown in FIG.2, little activity is noted in the ABP containing buffer diluent.Significant aPC reactivity is seen in the control sample diluted intothe buffer containing BSA which increases in the redox exposed sample.In addition, significant aPE, aCL and aPS reactivities appear subsequentto oxidation of the monoclonal and dilution into a buffer containingBSA.

Example 3

Example 1 was repeated, except that a CD 63 monoclonal antibody was usedas the monoclonal antibody instead of an anti-glycophorin A monoclonalantibody. The format of the ELISA testing of an IgG1 monoclonal antibodyto CD 63 was identical to that described in FIGS. 1 and 2. The bindingprofiles for the diluents containing ABP and BSA and the CD 63monoclonal antibody before EMF treatment (“control”) and after EMFtreatment (“redox”) are shown in FIG. 3. As shown in FIG. 3, oxidationof this monoclonal by EMF showed a single alteration, the appearance ofaPC in the BSA buffer sample.

Example 4

Example 1 was repeated, except that a monoclonal antibody to a plasmaprotein, beta2 glycoprotein I (β₂GP-I) was used as the monoclonalantibody instead of an anti-glycophorin A monoclonal antibody. Thebinding profiles for the diluents containing ABP and BSA and the β₂GP-Imonoclonal antibody before EMF treatment (“control”) and after EMFtreatment (“redox”) are shown in FIG. 4. Interesting aspects in FIG. 4are in the ABP containing buffer. β₂GP-I is not present in the BSAbuffer dilution, but is present in the ABP buffer diluent. β₂GP-I bindsto cardiolipin and is recognized by the monoclonal as shown in thecontrol sample. However, after redox oxidation by EMF, the monoclonalfails to recognize its β₂GP-I antigen. Thus oxidation has altered themonoclonals' binding site for β₂GP-I. However, after oxidation of thismonoclonal there is reactivity as an aPE antibody in the ABP containingbuffer diluent and aPC reactivity in the BSA containing diluent. Thus,there is simultaneous masking and unmasking of this monoclonals' aPLreactivities.

Example 5

Example 1 was repeated, except that an anti-factor VII monoclonalantibody was used as the monoclonal antibody instead of ananti-glycophorin A monoclonal antibody. The binding profiles for thediluents containing ABP and BSA and the anti-factor VII monoclonalantibody before EMF treatment (“control”) and after EMF treatment(“redox”) are shown in FIG. 5. As shown in FIG. 5, no redox alterationswere observed for aPL binding.

Example 6

Example 1 was repeated, except that a factor IX monoclonal antibody wasused as the monoclonal antibody instead of an anti-glycophorin Amonoclonal antibody. The binding profiles for the diluents containingABP and BSA and the factor XI monoclonal antibody before EMF treatment(“control”) and after EMF treatment (“redox”) are shown in FIG. 6. Asshown in FIG. 6, the IgG monoclonal antibody to factor IX is not altered(masked) by redox exposure as shown by the fact that factor IX, presentin the ABP buffer diluent, is equally recognizable by the antibody inthe control and redox samples. In the BSA diluent, because no factor IXis available, the control is negative but both aPE and aPC are unmaskedby EMF redox exposure.

Example 7

Example 1 was repeated, except that gamma 1 mouse control monoclonalantibody was used as the monoclonal antibody instead of ananti-glycophorin A monoclonal antibody. Additionally, an alternativeoxidative treatment was carried out using hemin under the conditionsdescribed above. In particular, hemin, 2.5 μl (15.15 mg/ml) was added to0.5 ml of the monoclonal antibody solution and incubated overnight at36° C. on a rocking platform. The binding profiles for the diluentscontaining ABP and BSA and the gamma 1 mouse control monoclonal antibodybefore either hemin or EMF treatment (“control”) and after “EMF” and“Hemin” treatment are shown in FIG. 7. FIG. 7 shows that that alterationof the monoclonal binding activities is affected by the oxidizing agent.In this example hemin unmasks aPS, aCL and aPE reactivities whereas EMFtreatment does not. Both hemin and EMF can unmask aPC which is mostnotable when the monoclonal is diluted into a buffer containing BSA. Themonoclonal antibody in this graph is sold commercially as an IgG isotypecontrol antibody and its antigen of record is hemocyanin, a protein notfound in the human repertoire. EMF exposure was the same as described inFIG. 1.

Example 8

Example 1 was repeated, except that a CD 44 monoclonal antibody was usedas the monoclonal antibody instead of an anti-glycophorin A monoclonalantibody. The binding profiles for the diluents containing ABP and BSAand the CD 44 monoclonal antibody before treatment (“control”) and after“Hemin” or “EMF” treatment are shown in FIG. 8. FIG. 8 shows that a thata commercially produced IgG1 monoclonal antibody to the antigen CD 44appears refractory to binding alterations when exposed to hemin and/orEMF as described in FIG. 5.

Example 9

Example 1 was repeated, except that an anti-platelet IIb-IIIa mousemonoclonal antibody was used as the monoclonal antibody instead of ananti-glycophorin A monoclonal antibody. The binding profiles for thediluents containing ABP and BSA and the CD 63 mouse monoclonal antibodybefore EMF treatment (“control”) and after EMF treatment (“redox”) areshown in FIG. 9. FIG. 9 shows that shows that a commercially producedIgG1 monoclonal to the platelet antigen IIb-IIIa becomes unmasked afterEMF treatment and appears as aPS and aCL in the presence of an ABPcontaining diluent, but not in a diluent containing BSA. The likelyexplanation for this observation is the presence of plasma antigens inthe ABP that can bind to PS and CL and that oxidation of this monoclonalalters its binding properties.

Example 10

Example 1 was repeated, except that a tailor made monoclonal antibody toa murine tumor cell line SP2/0 was used as the monoclonal antibodyinstead of an anti-glycophorin A monoclonal antibody. This monoclonalantibody (Mab) was produced in culture media wherein all the componentswere known and samples of the culture media were obtained before andafter growing the monoclonal to assure that all possible ELISA controlswere performed. The binding profiles for the ABP (FIG. 10A) and BSA(FIG. 10B) diluents of the SP2/0 monoclonal antibody before EMFtreatment (“Cntl”) and after EMF treatment (“EMF”) are shown. Inparticular, FIG. 10(A) is a graph showing the effect of EMF exposure ofa monoclonal antibody (Mab) produced to a murine tumor cell line SP2/0.All culture media components were known, thus oxidation by EMF exposureas shown in the above figure unmasks aPL reactivity of this monoclonal.In this figure the EMF+Mab on the left represents oxidation of theprotein-A purified monoclonal (1.46 mg/ml) diluted 1/10 into a bufferdiluent containing ABP and the ELISA was developed in substrate for only10 min. The remaining ELISA bar graphs represent substrate developmentfor 70 min. As anticipated, the media containing the oxidized monoclonalwithout concentration requires significantly more development time forpositive results to appear. FIG. 10(B) represents the identical samplesand conditions as depicted in FIG. 10A, but the diluent buffer in thisgraph contained BSA. The most striking observation in the BSA diluentbuffer when compared to the ABP diluent buffer is the increased signalof aPC. This may represent competition of binding between the aPC and aplasma protein in the ABP containing buffer or an antioxidant in the ABPcontaining buffer that is extraordinarily effective for inhibiting aPCunmasking.

Example 11

The identical anti-glycophorin A monoclonal antibody used in example 1was used to assess the effects of EMF oxidation on the monoclonalsrecognition of its red blood cell (RBC) membrane target antigen. Theexperimental design also used a 250 μl bubble of the monoclonal antibodysolution on parafilm, but a 3 μl sample was withdrawn at each 5 secondinterval for the first minute to test by flow cytometry for RBC binding.After 60 seconds, an additional EMF treatment was done for another 60seconds, uninterrupted (total EMF time 2 minutes). The results of thisexperiment are provided in FIG. 11. In particular, FIG. 11 demonstratesby using flow cytometric analysis of red blood cell (RBC) binding, theeffect of increasing the time of EMF oxidation on the identicalanti-glycophorin A monoclonal antibody (anti Gly A) preparation shown inFIG. 1. During the first minute of EMF exposure an aliquot of themonoclonal antibody was sampled for RBC binding every 5 seconds. A meanchannel shift (MCS) downward was observed starting at 401 before EMFapplication which decreased to a MCS of 223 at 60 seconds. A MCS from401 to 386 was observed at the 10 second interval and this was shown tounmask the aPL depicted in FIG. 1. A MCS difference of 15 would not beconsidered significant by flow cytometry operators. After the 60 secondaliquot was removed, an additional 60 seconds of EMF oxidation wasapplied to the remaining monoclonal antibody. The extended EMF oxidationtime totaled 120 seconds and as the line graph shows, the downward trendof binding to RBC was reversed and became an MCS of 359 which wasequivalent to the MCS after the initial 20 second exposure to EMFoxidation.

Example 12

The reversibility of altering the binding properties of monoclonalantibodies is shown in FIGS. 12A and 12B to be time and temperaturedependent. Stored at −80° C., the oxidized monoclonal antibodies retaintheir aPL reactivities. Storage of the monoclonal antibodies at 37° C.shows rapid loss of the aPL binding specificities and return to theirinitial non-oxidized aPL status. Another exposure to EMF oxidation,however, causes the monoclonals stored at 37° C. to again unmask. Inparticular, as shown in FIG. 12, 4-day storage of an oxidized monoclonalantibody to murine tumor SP2/0 at 37° C. shows a loss of aPL unmaskingactivity that is not observed at −80° C. The aPL reactivity can beunmasked again if the 37 degree sample is exposed to another EMFtreatment. Unfortunately, in this experiment, the aPE plate was droppedbefore its OD could be read and that's why the left panel histogram isblank for aPE. However, this experiment has been done several times withpolyclonal IgG and EMF and shows loss of aPE upon 37 degree storage andrecovery of aPE after a second EMF exposure.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is therefore to beunderstood that, within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

1. A method comprising: providing a solution comprising a monoclonalantibody, the monoclonal antibody having a binding specificity toward aspecific first antigen, exposing the composition to an oxidizing agentor an electric potential sufficient to effect an alteration of thebinding specificity of the monoclonal antibody, and screening themonoclonal antibody after the composition has been exposed to theoxidizing agent or electric potential to determine whether themonoclonal antibody has a binding affinity for other antigens other thanthe first antigen, wherein the monoclonal antibody is a therapeuticagent or a candidate therapeutic agent wherein the screening is carriedout to assess whether the monoclonal antibody has autoantibody activityunder oxidative conditions.
 2. The method of claim 1, wherein theoxidizing agent is hemin.
 3. The method of claim 1, wherein exposing ofthe composition to an electric potential is carried out by submersingelectrodes connected to a positive and negative terminal of a battery orelectric power source into the solution comprising the monoclonalantibody for a predetermined period of time.
 4. The method of claim 1,wherein the solution comprising the monoclonal antibody includes thecommercial manufacturers' buffers, TRIS buffer, phosphate bufferedsaline or culture media.
 5. A method of claim 1, further comprisingrecovering the monoclonal antibody having the altered bindingspecificity from the solution.